Reduced sensitivity of AChE has been reported as one of the major resistance mechanis

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Reduced sensitivity of AChE has been reported as one of the major resistance mechanis

Molecular and Kinetic Properties of Two Acetylcholinesterases from the Western Honey Bee, Apis mellifera
Western blot analysis revealed that AmAChE2 has most of catalytic activity rather than AmAChE1, further suggesting that AmAChE2 is responsible for synaptic transmission in A. mellifera, in contrast to most other insects. AmAChE2 was predominately expressed in the ganglia and head containing the central nervous system (CNS), while AmAChE1 was abundantly observed not only in the CNS but also in the peripheral nervous system/non-neuronal tissues. Acetylcholinesterase (AChE, EC 3.1.1.7) is a critical enzyme in the cholinergic synapses and neuromuscular junctions of both vertebrates and invertebrates that regulates the level of the neurotransmitter acetylcholine and terminates nerve impulses [1]. AChE is a key enzyme in the insect nervous system, in which the cholinergic system is essential [2], and is the target of organophosphate (OP) and carbamate (CB) insecticides. Reduced sensitivity of AChE has been reported as one of the major resistance mechanisms against OP and CB insecticides in many arthropods. http://www.plosone.org/article/info%...l.pone.0048838

Re: Reduced sensitivity of AChE has been reported as one of the major resistance mech

From biochemical point of view - weak paper. I my opinion, there is no structural data to support their modeling, thus - does not make much sense to me. This is a greatness and weakness of the PlosOne - there are great papers and very weak ones - reader need to do "pier reviewing" for any PlosOne paper. If something published, it is not necessary great paper...

Re: Reduced sensitivity of AChE has been reported as one of the major resistance mech

PLOSOne are peer reviewed. What would be enough structural data for you?

Materials and Methods Top

Insects

The colonies of the Western honey bee, A. mellifera, that were used as sources for experimental specimens were maintained at Seoul National University. According to their behaviors and ages [37], we collected forager bees, which were older than 3 weeks of age and returned to the hive with clearly visible pollen loads on their hind legs. The collected honey bees were frozen directly with liquid nitrogen and stored at −75°C until protein extraction.

Protein Sample Preparation

Soluble proteins were extracted from the forager heads with 0.1 M Tris-HCl (pH 7.8) buffer, while membrane-bound proteins were extracted with the same buffer supplemented with 0.5% Triton X-100. To observe the tissue distribution of AmAChE1 and AmAChE2, protein samples were prepared from six various tissues (ganglia, head, thorax, abdomen, leg, and gut) of forager bees with buffer containing Triton X-100. Proteins from various samples were extracted with an appropriate amount of buffer using a micro tissue grinder (Radnoti, Monrovia, CA, USA). The homogenates were centrifuged at 12,000×g for 15 min at 4°C. The supernatant was filtered through glasswool to remove excess lipid and stored at −75°C until use.

Antibody Generation, PAGE and Western blotting

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), native-PAGE, Western blotting and AChE activity staining were conducted as previously described with some modifications [22]. Electrophoresis was performed with a vertical electrophoresis unit (Novex® mini cell, Invitrogen, Carlsbad, CA, USA). Protein preparations from various tissues (20 µg) were separated by native-PAGE gel (7.5%) in triplicate at 120 V for 90 min in a cold chamber with a continuous Tris-glycine buffer system. The gel and running buffers contained 0.5% Triton X-100 (v/v). Following native-PAGE, one set of gels was stained for AChE activity according to previously described methods [38], while the remaining two sets of gels were analyzed by Western blotting as described below.

To determine the multimer formation of AmAChEs, 20 µg of protein samples extracted from forager heads were treated with or without 14 mM ß-mercaptoethanol and separated by SDS-PAGE (4–12% gradient gel). To investigate the anchor properties of AmAChEs, proteins extracted from forager heads (20 µg) were incubated with 0.13 U of phospholipase C (PIPLC) for 20 min at 20°C. Non-treated control samples were incubated in the absence of PIPLC. After digestion, protein samples were separated on a native-PAGE gel in triplicate. After PAGE, one set of gels was stained for AChE activity, while the remaining two sets of gels were analyzed by Western blotting; one set of gels was analyzed with an AChE1-specific antibody, while the other was analyzed with an AChE2-specific antibody, as described below.

Proteins separated on the gels were transferred to Hybond-N nitrocellulose membranes (GE Healthcare, Pittsburgh, PA, USA) by electroblotting. After blocking in PBS buffer containing 0.1% Tween-20 (PBST) and 5% fat-free dry milk for 1 h at room temperature, the nitrocellulose membrane sheets were incubated for 3 h at room temperature or overnight at 4°C with primary antibodies (anti-AChE1 or anti-AChE2) [22]. Anti-AChE1 and anti-AChE2 polyclonal antibodies were generated as described previously [22]. The membranes were then incubated with horseradish-peroxidase conjugated anti-rabbit IgG secondary antibody (Pierce Bio-Technology, Rockford, IL, USA) for 1 h. The antigen-antibody complex on the bands was visualized with a chemiluminescence kit according to the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

In vitro Expression of AmAChE1 and AmAChE2 with a Baculovirus Expression System

Total RNA was extracted from forager heads with TRI reagent (MRC, Cincinnati, OH, USA) as described by the manufacturer. Following extraction, the total RNA was treated with DNaseI (TAKARA Korea Biomedical Inc., Seoul, Korea) at 37°C for 30 min and concentrated with 3 M sodium acetate. First strand cDNA was synthesized from the DNaseI-treated total RNA with Superscript III reverse transcriptase (Invitrogen) at 55°C for 1 h by priming with oligo dT, and the RNA strand was then removed by incubation with RNase H (Invitrogen) at 37°C for 20 min. The complete cDNA fragments encoding A. mellifera ace1 and ace2 (AmAce1 and AmAce2, respective GenBank accession numbers XM393751 and AF213012) were amplified by Advantage Taq (Clontech, Palo Alto, CA, USA) with gene-specific primers (Table S1) and directly cloned into the pGEM®-T easy vector (Promega, Madison, MU, USA). Partial fragments of AmAce1 and AmAce2 with truncated C-terminal hydrophobic regions were amplified by ExTaq (Takara, Japan) at 95°C for 2 min, (95°C for 30 s, 65°C for 30 s, 72°C for 2 min) × 5 cycles, (95°C for 30 s, 60°C for 30 s, 72°C for 2 min) × 30 cycles and 72°C for 2 min with gene-specific primers containing restriction enzyme sites (Table S1) from respective full ORF clones. The amplified AmAce1 and AmAce2 DNA fragments were digested with XbaI and SacI (Koschem, Korea) and inserted into pBacPAK8 (Clontech) that had been digested with the same restriction endonucleases. Recombinant baculoviruses expressing AmAChE1 and AmAChE2 in SF9 cells were generated as described previously [22]. Virus-infected cells were incubated for 84 h at 27°C. Protein samples were collected by centrifugation and concentrated with an Ultra Amicon YM-30 (Millipore, Bedford, MA, USA). Protein concentrations were determined by the Bradford method with bovine serum albumin as the standard protein [39]; the proteins were then stored at −75°C until use.

Kinetics and Inhibition of AmAChEs

The enzyme assay for AmAChEs expressed in Sf9 cells was performed with 8 different concentrations (0.05 to 1 mM) of acetylthiocholine iodide (ATChI) and butyrylthiocholine iodine (BTChI) according to previously described methods with some modifications [40], [41]. To measure enzyme kinetics, 15 µl of culture supernatant containing 5 µg of one of the two AmAChEs as enzyme sources was added to each well containing 85 µl of substrate mixture in the presence of 0.4 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); the wild type virus was used as a blank control. The reaction was monitored at 412 nm for 5 min with 10-sec intervals with a Soft Max® Pro5 microplate reader (Molecular Devices, Menlo Park, CA) at 30°C. Michaelis-Menten constants (Km) and maximal velocity (Vmax) values for each substrate were determined by Lineweaver-Burk plot.

AmAChE inhibition was assayed at 7 different concentrations (10−9 to 10−3 M) of each of three inhibitors (BW284C51, eserine and Iso-OMPA), three organophosphates (DDVP, malaoxon and paraoxon) and four carbamates (aldicarb, carbaryl, carbofuran and propoxur), according to previously described methods with some modifications [42]. Due to high sensitivity of AmAChEs to chlorpyrifos-oxon, 10−12 to 10−6 M of chlorpyrifos-oxon was used for inhibition assay. Five µg of each AmAChE as enzyme source was added to each well containing the substrate mixture of 1 mM ATChI and 0.4 mM DTNB and various concentrations of inhibitors to initiate enzyme reaction. Upon initiation of reaction, AmAChE activities were recorded at 412 nm for 3 min with 10-sec intervals using a microplate reader as above. To investigate the scavenger effects of AmAChE1 on the inhibition of AmAChE2, an AmAChE mixture was inhibited with 7 different concentrations of each of two OP (chlorpyrifos -oxon and malaoxon) and CB (carbofuran and propoxur) according to previously described methods with some modification [43]; the molar ratio of AmAChE1:AmAChE2 was 2:1 (4:2 µg) based on the their expressed protein levels in forager bees. The inhibitors were pre-incubated with AmAChE1 for 30 min at 30°C, and AmAChE2 was then added. The same molar amount of BSA instead of AmAChE1 was used as a control. AmAChE activities in the presence of 1 mM ATChI, 0.4 mM DTNB and various concentrations of inhibitors were recorded as above. Five consecutive concentrations of each inhibitor exhibiting a relatively linear range of inhibition were selected for the calculation of median inhibition concentration (IC50). The IC50 for each inhibitor was determined based on log-concentration versus probit (% inhibition) regression analysis using SPSS for Windows version 20.0 K (SPSS Inc., Chicago, IL).

3D Structure Modeling, Hydrophobicity and GPI-anchor Prediction

The 3D structure analyses of AmAChE1 and AmAChE2 were performed with the Automated Comparative Protein Modeling Server of SWISS-MODEL (http://swissmodel.expasy.org/) using human BChE and D. melanogaster AChE as the respective templates. Structure comparisons between AmAChEs were performed with UCSF Chimera MatchMaker ver. 1.4 (University of California, CA). The models were visualized and modified with Swiss PDB viewer 4.0.1 (Swiss Institute of Bioinformatics, Lausanne, Switzerland). The hydrophobicities of AmAChE1 and AmAChE2 were predicted with ProtScale of the ExPASy Proteomics Server (http://expasy.org/cgi-bin/protscale.pl). The potential GPI-anchor sequences of two AmAChEs were predicted with GPI-SOM of the ExPASy Proteomics Server (http://gpi.unibe.ch/).

Results Top

Expression Patterns of AmAChEs in Various Tissues

To determine the tissue-specific expression profiles of two AmAChEs, native-PAGE was performed on proteins extracted from six tissues (thoracic ganglia, head, thorax, abdomen, legs and gut) of forager bees, and their AChE activities were visualized by activity staining (Fig. 1B). The AmAChEs were concentrated in the ganglia and heads containing the central nervous system (CNS) (band ‘a’), but little activity was detected in the peripheral nervous system (PNS) such as in the thorax, abdomen, legs and gut.

Figure 1. Tissue distribution of AmAChE1 and AmAChE2 as assessed by native polyacrylamide gel electrophoresis and Western blot analysis.

Protein samples (20 µg) from various tissues were loaded onto a 7.5% polyacrylamide gel and run for 90 min at 120 V. After electrophoresis, one gel was stained for activity with acetylthiocholine iodide as a substrate (B). The other gels were analyzed by Western blot with anti-AChE1 (A) or anti-AChE2 (C) polyclonal antibodies.
doi:10.1371/journal.pone.0048838.g001

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Western blot analysis revealed that the main AChE activity is actually associated with AmAChE2 (Fig. 1C, band ‘a’), and AmAChE2 was more predominantly expressed in the CNS than in other tissues. By contrast, although AmAChE1 activity was faintly detected in ganglia (Fig. 1B, band ‘b’), AmAChE1 was not detectable in other tissues. Nevertheless, AmAChE1 seemed to be abundantly expressed not only in the CNS but also in the PNS such as in the thorax, abdomen and legs, as judged by Western blot analysis (Fig. 1A, band ‘b’).

Molecular Characterization of AmAChEs

Crude protein was extracted from forager heads with 0.1 M Tris-HCl buffer in the presence or absence of Triton X-100 to determine the molecular formations and soluble nature of AmAChE1 and AmAChE2. When the protein was extracted with Triton X-100-containing buffer, both AmAChE1 and AmAChE2 were strongly detected (Fig. 2A, see the Triton X-100 (+) lanes). In the absence of Triton X-100, the AmAChE1 band was still clearly observed, while AmAChE2 was faintly detected (Fig. 2A, see the Triton X-100 (−) lanes), suggesting the membrane-anchored nature of AmAChE2. To confirm the membrane-anchored properties of AmAChEs, AmAChEs extracted from forager heads were digested with PIPLC. After PIPLC treatment, no molecular changes were observed in AmAChE1, whereas amphiphilic AmAChE2 was completely converted to the hydrophilic form (Fig. 2B). This result indicates that AmAChE2 is associated with the membrane via a GPI-anchor, while AmAChE1 is present in a soluble form due to the absence of a GPI-anchor, as indicated by PIPLC treatment as well as protein extraction in the absence of Triton X-100.

Figure 2. Molecular characterization of AmAChE1 and AmAChE2 by polyacrylamide gel electrophoresis and Western blot analysis.

Protein samples were extracted from the heads of forager honey bees with 0.1 M Tris-HCl buffer in the presence or absence of 0.5% Triton X-100 to determine the soluble nature of the AmAChEs (A). To investigate the GPI-anchor properties of the AmAChEs, protein samples were treated with PIPLC (B). Protein samples were mixed with or without β-mercaptoethanol and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis to determine the multimer formation of AmAChE1 and AmAChE2 (C).
doi:10.1371/journal.pone.0048838.g002

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The molecular masses of AmAChEs were analyzed by SDS-PAGE in the presence or absence of β-mercaptoethanol reduction (Fig. 2C). Under non-reducing conditions, bands of approximately 185 kDa (band ‘a’) and 140 kDa (band ‘e’) were strongly visualized as putative dimers, while bands of 88 kDa (band ‘b’) and 79 kDa (band ‘f’) were faintly detected as putative monomers by the respective AmAChE1- and AChE2-specific antibodies (see reduction (-) lanes). In addition to the putative dimeric form, a band of approximately 50 kDa representing cleaved monomer (band ‘g’) was also clearly detected for AmAChE2. This finding suggests that dimers are the predominant forms of both AmAChEs under native conditions and that the cleaved monomer is also abundantly present in AmAChE2. After reduction with β-mercaptoethanol, the relative quantities of the other forms, such as the monomers (88 kDa band ‘b’ for AmAChE1 versus 70 kDa band ‘f’ for AmAChE2) and cleaved forms (48 kDa band ‘c’ and 40 kDa band ‘d’ for AmAChE1 vs. 50 kDa band ‘g’ for AmAChE2), increased, while the dimeric forms of both AmAChEs disappeared, supporting the presence of a disulfide bond in the dimer conformation. As indicated by the amino acid sequence alignment with Drosophila and German cockroach AChEs (Fig. S1B), the presence of cysteine at the C-terminal region of several insect AChEs also supports the role of a disulfide bond connection in dimer formation for both AmAChE1 and AmAChE2.

Kinetic Properties of AmAChEs

As judged by Western blotting with AChE1- and AChE2-specific antibodies, two types of AmAChEs were successfully expressed with the recombinant baculovirus expression system. In vitro expressed AmAChE2 demonstrated strong AChE activity, whereas AmAChE1 activity was barely detectable, as observed in the protein samples from the heads of forager honey bees (Fig. S2A). In addition, the estimated molecular masses of both AmAChEs (approximately 70 kDa) were confirmed by Western blotting following SDS-PAGE (Fig. S2B).

Two cholinesterase substrates, ATChI and BTChI, were used to study the kinetic properties of the two AmAChEs. The Km and Vmax values were calculated by double-reciprocal plots (Fig. S3). The kinetic parameters for the two substrates are presented in Table 1. AmAChE1 and AmAChE2 exhibited 3.8- and 4-fold higher catalytic efficiency [Maximal velocity/Michaelis-Menten constants (Vmax/Km)], respectively, toward ATChI than BTChI, confirming the typical substrate specificities of AChEs. In a cross-enzyme comparison of AmAChE1 and AmAChE2, AmAChE2 showed lower Km values than AmAChE1 (1.28- and 12.5-fold for ATChI and BTChI, respectively), demonstrating higher substrate affinity compared to AmAChE1. AmAChE2 also exhibited approximately 200-fold higher Vmax values for both ATChI and BTChI than did AmAChE1. Taken together, AmAChE2 exhibited approximately 2,500- and 2,400-fold higher catalytic efficiencies toward ATChI and BTChI, respectively, than did AmAChE1, indicating that AmAChE2 is a much more efficient enzyme than AmAChE1. The ratio Vmax(BTChI)/Vmax(ATChI) was 0.51 and 0.5 for AmAChE1 and AmAChE2, respectively, indicating that the substrate spectrums of AmAChE1 and AmAChE2 were similar.

Table 1. Kinetic properties of recombinant two AmAChEs in hydrolyzing various substrates*.
doi:10.1371/journal.pone.0048838.t001

The inhibitory properties of AmAChEs were determined with various concentrations of three reversible cholinesterase-specific inhibitors, four OPs and four CBs (Fig. S4 and Table 2). Both AmAChEs were effectively inhibited by BW284C51 and eserine but not by Iso-OMPA, a BChE-specific inhibitor, suggesting that both enzymes retain typical features of AChEs [44]. BW284C51 similarly inhibited both AmAChEs, while AmAChE2 was 16-fold more sensitive to eserine than was AmAChE2, as judged by IC50 values. In the inhibition assay with OPs and CBs, AmAChE2 was, in general, much more sensitive to these insecticides than was AmAChE1 (Table 2). As indicated by the IC50 values of the OPs, AmAChE2 was approximately 4-, 7-, 3- and 45-fold more sensitive to chlorpyrifos, DDVP, malaoxon and paraoxon, respectively. The CBs also inhibited AmAChE2 more effectively than did AmAChE1 (3,500-, 19- and 3-fold for carbaryl, carbofuran and propoxur, respectively), whereas neither AmAChE was inhibited by aldicarb. In a cross-inhibitor comparison of OPs and CBs, OPs generally exhibited much lower IC50 values than CBs, suggesting that OPs are more efficient inhibitors of both AmAChE1 and AmAChE2 than CBs.

Table 2. IC50 (M) values of different inhibitors of two recombinant AmAChEs*.
doi:10.1371/journal.pone.0048838.t002

To examine the physiological function of AmAChE1, the inhibition rates of two OPs and two CBs against AmAChE2, the major catalytic enzyme in the honey bee, were measured in the presence or absence of AmAChE1 (Fig. 3). As judged by the IC50 values (Table S2), the overall inhibition was reduced 2.3- to 4.5-fold when inhibitors were pre-incubated with AmAChE1 and then added to AmAChE2 compared to pre-incubation with BSA, suggesting that one of the physiological functions of AmAChE1 may be chemical defense against xenobiotics, perhaps by sequestering inhibitors.

Figure 3. Inhibition of AmAChE2 by two organophosphates (A) and two carbamates (B) in the presence (○) or absence of AmAChE1 (

•). Inhibitors were pre-incubated with AmAChE1 for 30 min at 30°C, and AmAChE2 was then added. The same molar amount of BSA in place of AmAChE1 was used as a control.
doi:10.1371/journal.pone.0048838.g003

The 3D structures of AmAChE1 and AmAChE2 were predicted with Swiss Model using the structures of human BChE and D. melanogaster AChE as the respective templates (Fig. 4). In the structure comparison between AmAChE1 and AmAChE2, most of the α-helix and β-stranded sheets were highly overlapped, demonstrating that the overall structures of the two AmAChEs were similarly folded (Fig. 4A). When the catalytic gorge structures of the two AmAChEs were merged, the choline-binding site (W148) and the catalytic triad (S263, E389 and H504) of AmAChE1 closely overlapped those of AmAChE2 (Fig. 4B). However, the shape of the peripheral anionic site (PAS) changed because of differences in two amino acid residues: Y185 in AmAChE1 versus M170 in AmAChE2 and C350 in AmAChE1 versus L343 in AmAChE2. In addition, the angles of the choline-binding site (W344/336) at the entrance to the active gorge in AmAChE1 and AmAChE2 differed by approximately 90°, suggesting that the differences in the PAS conformation may be responsible for the differences in the substrate and inhibition kinetics of AmAChE1 and AmAChE2.

The superimposed 3D structures (A) and zoom-in views (B) of the two AmAChEs were compared. The positions of amino acid residues involved in the formation of the catalytic triads, choline-binding sites and peripheral anionic sites are indicated (AmAChE1/AmAChE2) (B).
doi:10.1371/journal.pone.0048838.g004

Re: Reduced sensitivity of AChE has been reported as one of the major resistance mech

Originally Posted by Acebird

Is the findings of this paper concluding that insects build up resistance to insecticides?

The bottom line for beekeepers for sure Ace. My thesis for graduate school researched and reported on how few research papers actually had impact or application to someone providing health care. Basic research is important. I'm more interested in how research applies to what you and I do every day in the bee yard. The jargon in the cited paper makes understanding possible for just a few who speak that language.